ES2333845T3 - METHOD AND COMPOSITION FOR THE INHIBITION OF ANGIOGENESIS. - Google Patents

Abstract

The invention describes methods for inhibiting angiogenesis in a tissue by administering an antagonist that specifically binds to a proteolyzed or denatured collagen but not to native triple helical forms of the collagen. Antagonists of the invention can target, for example, denatured collagens type-I, type-II, type-III, type-IV, type-V and combinations thereof. Methods utilizing such antagonists for therapeutic treatment of tumor growth, tumor metastasis or of restenosis also are described, as are methods to use such antagonists as diagnostic markers of angiogenesis is normal or diseased tissues both in vivo and ex vivo. Antagonists include monoclonal antibodies referred to as HUI77, HUIV26, and XL313.

Description

Method and composition for the inhibition of angiogenesis

Field of the Invention

The invention generally relates to medical field and refers specifically to methods and compositions to inhibit angiogenesis in a tissue or detect angiogenesis using antagonists of denatured forms or collagen proteolytes that include, but are not limited to, types I, II, III, IV and V. WO 94/14070 describes a method to determine cartilage degradation in a sample biological using a monoclonal antibody. This method is described. as one that measures the amount of uncoiled collagen present in a biological sample by binding uncoiled collagen with a monoclonal antibody.

Background

Tumor growth and metastasis affect a large number of people every year. In fact, it is estimated that much more than 600,000 new cases of cancer will be diagnosed in the United States next year alone (Varner, JA, Brooks, P. C and Cheresh, DA (1995) Cell Adh. Commun. 3, 367- 374). To a large extent, numerous studies have suggested that the growth of all solid tumors requires growth of new blood vessels for the continued expansion of tumors beyond a minimum size (Varner et al . 1995; Blood, CH and Zetter, BR ( 1990) Biochim. Biophys. Acta. 1032: 89-118; Weidner, N. et al . (1992) J. Natl. Cancer Inst. 84: 1875-1887; Weidner, N. et al . (1991). Engl. J. Med. 324: 1-7; Brooks, PC et al . (1995) J. Clin. Invest. 96: 1815-1822; Brooks, PC et al . (1994) Cell 79: 1157-1164; Brooks , PC et al . (1996) Cell 85, 683-693; Brooks, PC et al . (1998) Cell 92: 391-400. Significantly, a wide diversity of other human diseases are also characterized by unregulated development of blood vessels, including eye diseases such as macular degeneration and diabetic retinopathy.In addition, numerous inflammatory diseases are also associated with uncontrolled neovascularization such as artrit is and psoriasis (Varner et al. nineteen ninety five). Angiogenesis is the physiological process by which new blood vessels develop from pre-existing vessels (Varner et al . 1995; Blood and Zetter 1990; Weidner et al. 1992). This complex process requires the cooperation of a variety of molecules that include growth factors, cell adhesion receptors, matrix degrading enzymes and extracellular matrix components (Varner et al . 1995; Blood and Zetter 1990; Weidner et al. 1992 ). Therefore, therapies designed to block angiogenesis can significantly affect the growth of solid tumors. In fact, clear evidence has been provided that blocking tumor neovascularization can significantly inhibit tumor growth in various animal models and human clinical data are also beginning to support this argument (Varner, JA, Brooks, PC and Cheresh , DA (1995) Cell Adh. Commun. 3, 367-374). To a large extent, numerous studies have suggested that the growth of all solid tumors requires the growth of new blood vessels for the continued expansion of tumors beyond a minimum size (Varner et al. 1995; Blood and Zetter 1990; Weidner et al. 1992; Weidner et al . 1991; Brooks et al . 1995; Brooks et al. 1994; Brooks et al . 1997).

To this end, many researchers have focused their anti-angiogenic strategies towards growth factors and cytokines that initiate angiogenesis (Varner et al . 1995; Blood and Zetter 1990; Weidner et al . 1992; Weidner et al. 1991; Brooks et al 1995; Brooks et al . 1994; Brooks et al . 1997). However, there are a large number of different growth factors and cytokines that have the ability to stimulate angiogenesis. The therapeutic benefit of blocking a single cytokine may have only limited benefit due to its redundancy. However, little attention has been directed towards other anti-angiogenic targets. Recent studies have suggested that angiogenesis requires proteolytic remodeling of the extracellular matrix (ECM) that surrounds blood vessels to provide a microenvironment that leads to the development of new blood vessels ( Varner et al. 1995; Blood and Zetter 1990; Weidner et al . 1992; Weidner et al . 1991; Brooks et al . 1995; Brooks et al . 1994; Brooks et al. 1997). Extracellular matrix protein collagen represents more than 25% of the total protein mass in animals and most of the protein within the ECM. Collagen is a multi-chain fibrous triple helix protein that exists in numerous forms (Olsen, BR (1995) Curr. Opin. Cell Biol. 7, 720-727; Van der Rest, M. and Garrone, R. (1991) FASEB 5, 2814-2823). At least 18 genetically distinct types of collagen have been identified, many of which have different tissue distributions and functions (Olsen 1995; Van der Rest and Garrone 1991). Type I collagen is the most abundant type of collagen in the extracellular matrix. It has been shown that type I, type III collagen, type IV collagen and type V collagen are associated with all pre-existing blood vessels in vivo. Type I and type IV collagens are composed of major chains called α1 (I) and α2 (I) and α1 (IV) and α2 (IV) respectively. The mature collagen molecule is composed of two α1 chains and one α2 chain wound in a triple helix. In vivo, collagen is normally in the form of mature triple helix. Denaturation of the native three-dimensional structure of the mature triple helix collagen can expose cryptic regulatory regions that control angiogenesis. The antagonism of these cryptic regulatory regions could provide an unrecognized means for diagnosis and inhibition of angiogenesis.

It has been proposed that inhibition of angiogenesis would be a useful therapy to restrict tumor growth. Inhibition of angiogenesis has been proposed by (1) inhibition of release of "angiogenic molecules" such as βFGF (fibroblast growth factor), (2) neutralization of angiogenic molecules, such as by the use of anti-\ antibodies. betaFGF and (3) inhibition of the response of endothelial cells to angiogenic stimuli. The latter strategy has gained attention and Folkman et al ., Cancer Biology, 3: 89-96 (1992), have described several inhibitors of endothelial cell response, including collagenase inhibitors, basal membrane renewal inhibitors, angiostatic steroids, Angiogenesis inhibitors obtained from fungi, platelet factor 4, thrombospondine, arthritis drugs, such as D-penicillamine and gold thiomalate, vitamin D3 analogues, alpha interferon and the like that could be used to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis, see Blood and Zetter 1990; Moses et al . (1990) Science 248: 1408-1410; Ingber et al . (1988) Lab. Invest., 59: 44-51; and U.S. Patent Nos. 5,092,885, 5,112,946, 5,192,744 and 5,202,352. None of the angiogenesis inhibitors described in the previous references are directed to denatured or proteolyzed collagen.

Summary

The present invention provides the use of denatured or proteolyzed collagen antibody antagonists for the manufacture of a composition that can inhibit angiogenesis. Antagonists specifically bind to a denatured or proteolyzed collagen, but bind with substantially reduced affinity to native forms of the same collagen. The antagonists may be specific for any denatured collagen, including denatured type I collagen, denatured type II collagen, denatured type III collagen, denatured type IV collagen or denatured type V collagen or combinations thereof. For example, in one embodiment, an antagonist is specific for denatured type I collagen with respect to native triple helix type I collagen but binds with substantially reduced affinity to other denatured collagen, such as type IV collagen. In another embodiment, an antagonist is specific for denatured type IV collagen. An antagonist can also be specific for type I, II, III, IV and V collagen
denatured

An antagonist of the invention is an antibody or a functional fragment thereof, which immunoreacts with denatured collagen but that immunoreacts in a range substantially less with the native form of collagen. The antibodies can be monoclonal. An antagonist could also be a polypeptide or a peptide with specificity for a collagen denatured, but not by a native form of collagen. The antagonists could also be non-peptide compounds such as a small organic molecule or an oligonucleotide.

The tissue to be treated can be any tissue in whichever inhibition of angiogenesis is desirable, such as diseased tissue in which the neo-vascularization: exemplary tissues include inflamed tissue, solid tumors, metastases, tissues that They experience restenosis and the like.

The invention also provides methods for detecting angiogenesis in a tissue by contacting an antagonist of the invention with the tissue. Such methods are suitable for use both ex vivo and in vivo.

A use is also provided for detecting tumor tissue, metastasis and tumor invasion in a tissue by contacting an antagonist of the invention with an tissue ex vivo or in vivo.

The invention also provides methods for select antagonists that specifically bind to a collagen or denatured collagens, but that bind with affinity substantially reduced to the native form of the collagen or the collagens and can inhibit angiogenesis.

Brief description of the figures

Figure 1 illustrates the reactivity of the Mab HUI77 with components of the extracellular matrix in solid phase ELISA. Microtiter plates (96 wells) were coated with extracellular matrix components that included type collagen Native type I and type IV, denatured type I and type IV collagen, vitronectin, fibronectin and fibrinogen, each at a concentration of 10 micrograms (mug) per milliliter (ml). The microtiter plate wells were blocked with 1% BSA in PBS for 1 hour at 37 ° C. Mab HUI77 was added to the wells at a concentration of 1 µg / ml and allowed to incubate for 2 hours at 37 ° C. After incubation, immunoreactivity was detected by incubation with goat secondary antibody anti-mouse labeled with peroxidase. The immunoreactivity was measured with an ELISA plate reader at 490 nm using c-phenylenediamine as a substrate. Col-I (native triple helix type I collagen). Denatured Col-I (Type I Collagen Denatured). Col-IV (type IV collagen of native triple helix). Denatured Col-IV (denatured collagen, types. VN (Vitronectin). FN (Fibronectin). FB (fibrinogen). Data bars represent The Optical Density (D.O.) means ± standard deviations of triplicate wells.

Figure 2 demonstrates the reactivity of Mab HUI77 with genetically distinct forms of collagen. They were coated  microtiter plates with different forms of collagen at a concentration of 10 µg / ml. The wells of the plates microtiter were blocked with 1% BSA in PBS for 1 hour at 37 ° C Immunoreactivity was detected by incubation with secondary goat anti-mouse labeled antibody with peroxidase Immunoreactivity was measured by determining the optical density with an ELISA plate reader at 490 nm. Col-I, Collagen I triple helix. Col-I Den; Denatured Collagen I Col-II; Collagen II triple helix. Col-II Den; Denatured Collagen II Col-III; Collagen III triple helix. Col-III Den; Denatured Collagen III Col-IV; Collagen IV triple helix. Col-IV Den; Denatured Collagen IV. Col-V; Collagen V triple helix. Col-V Den; Denatured Collagen V The bars of data represent the D.O. mean ± standard well deviation in triplicate.

Figure 3 shows that Mab HUI77 identifies the denatured collagen that surrounds human melanoma tumors in vivo. The generation and localization of denatured collagen in vivo was studied by indirect immunofluorescence in frozen tissue sections of human melanomas of both the human-mouse chimeric model and human tumor biopsies. Frozen tissue sections of human melanomas were fixed in acetone, blocked with 1% BSA and co-stained with Mab HUI77 and polyclonal antibody directed against α-nu integrin expressed in human melanoma cells. Antibody binding was detected by incubation with secondary goat anti-mouse antibody conjugated to FITC and goat anti-rabbit conjugated with rhodamine. The left panels indicate the human melanoma tumor biopsy (630X). The right panels indicate the M21 human melanoma cell line developed in full-thickness human skin (200X). Red indicates the expression of integrin [alpha] and green indicates expression of denatured collagen. Yellow indicates the co-localization of α-γ integrins and denatured collagen.

Figure 4 shows that Mab HUI77 identifies the denatured collagen that surrounds blood vessels associated with human melanoma tumor. The generation and localization of denatured collagen in vivo was studied by indirect immunofluorescence in frozen tissue sections of human melanoma tumor biopsies. Frozen tissue sections of human melanomas were fixed in acetone, blocked with 1% BSA and co-stained with Mab HUI77 and polyclonal antibody directed against HIV factor, a known marker of blood vessels. Antibody binding was detected by incubation with goat anti-mouse secondary antibodies conjugated to fluorescein isothiocyanate (FITC) and goat anti-rabbit conjugated with rhodamine. The left panel shows biopsy staining of human melanoma tumor for factor VIII (Red) that outlines a tumor blood vessel. The right panel shows the denatured (Green) collagen that surrounds the blood vessel associated with human tumor.

Figure 5 demonstrates the effects of Mab HUI77 on the adhesion of human endothelial cells. Plates microtiter (96 wells) were coated with type I collagen or type IV native or denatured at a concentration of 10 \ mug / ml. Wells of the microtiter plate were blocked with 1% BSA in PBS for 1 hour at 37 ° C. Then, it was left that human endothelial cells (HUVEC) bind to the wells coated in the presence or absence of purified Mab HUI77 (50 µg / ml) or a control antibody corresponding to isotype a a concentration of 50 µg / ml for 30 minutes. No cells bound were removed by washing and the bound cells were stained with crystal violet Cell adhesion was quantified by measuring the optical density (D.O.) of the dye eluted at 600 nm. The Bars of data represent the D.O. mean ± standard well deviation in triplicate. The data is represented as a percentage of control.

Figure 6 demonstrates the effects of Mab HUI77 on the migration of human endothelial cells. They were coated Transwell migration chamber membranes with type I collagen or Type IV denatured at a concentration of 25 µg / ml. Be let human endothelial cells (HUVEC) migrate in the presence or absence of purified Mab HUI77 or a control antibody corresponding to isotype (100 µg / ml) for a total of 6 hours. The cells that remained on the upper side of the membrane were removed and the cells that had migrated to the side Lower membrane stained with crystal violet. The migration Cellular was quantified by measuring the optical density (D.O.) of dye eluted at 600 nm with a microplate reader. The Bars of  data represent the D.O. mean ± standard well deviation in triplicate. Data are expressed as a percentage of control.

Figure 7 demonstrates the effects of systemic administration of purified Mab HUI77 on angiogenesis in vivo. Filter discs saturated with βFGF were placed on the Chorioallantoid (CAM) membranes of 10-day-old chicken embryos. Twenty-four hours later, the embryos received a single intravenous injection with 20 µg of Mab HUI77 or a control. At the end of a 3 day incubation period, the filter discs and surrounding CAM tissues were removed and the angiogenesis was quantified by counting the number of branching points of blood vessels within the area of the filter disc. Examples of CAM tissue from a typical experiment are shown.

Figure 8 shows the quantification of the Angiogenesis experiments with the Mab HUI77. The bars of data represent the mean ± typical errors of 5 to 10 embryos by condition The angiogenesis index is equal to the number of branching points of experimentally treated embryos less the number of CAM branching points in the absence of βFGF.

Figure 9 shows the effects of systemic administration of purified Mab HUI77 on tumor growth in vivo. TO). CS-1 melanoma tumor cells (5 x 10 6) were inoculated into CAMs of 10-day-old chicken embryos. Twenty-four hours later, the embryos received a single intravenous injection with 100 µg of purified Mab HUI77 or a control. The embryos were allowed to incubate for a total of 7 days. At the end of the 7 day incubation period, the resulting tumors were removed and wet weights determined. The photographs show representative tumors taken from embryos treated with or without Mab HUI77.

Figure 10 shows weight quantification in wet tumors. Data bars represent the average ± typical errors of tumor weights from 5 to 12 embryos by condition.

Figure 11 demonstrates the reactivity of Mab HUIV26 with extracellular matrix components in phase ELISA solid. Microtiter plates were coated with components of the extracellular matrix, each at a concentration of 25 \ mug / ml. TO). Mab HUIV26 was added at a concentration of 1 ? / ml, followed 1 hour after goat IgG peroxidase labeled anti-mouse. They prepared type 1 collagen and type IV collagen denatured by boil for 15 minutes before coating the plates. Everybody the data was corrected for any non-specific union of secondary antibody Data bars represent density optical (D.O.) mean ± standard deviations of wells per triplicate. B). The microtiter plates were coated with Type IV triple helix collagen at 25 µg / ml. Medium was added conditioning for concentrated HUVEC (20x) to the wells in presence or absence of EDTA, Aprotinin or both and allowed to incubate for 1, 6 and 24 hours. Then the plates were washed, they were blocked and incubated with Mab HUIV26 or control antibody. All data was corrected for secondary antibody binding. not specific Data bars represent optical density (D.O.) mean ± standard deviations of wells in triplicate. The following abbreviations are used in the figure, Col-I, type I collagen; Col-IV, type IV collagen.

Figure 12 demonstrates that Mab HUIV26 identifies denatured collagen IV that surrounds Angiogenic Blood Vessels within the Chorioallantoid Chicken Membrane (CAM). The generation and localization of denatured collagen IV in vivo was studied by indirect immunofluorescence in frozen tissue sections of CAM tissue of both bFGF-induced and tumor-induced angiogenesis. Frozen CAM tissue sections were fixed in acetone, blocked with 1% BSA and co-stained with Mab HUIV26 and polyclonal antibodies directed against collagen degrading enzyme IV MMP-2 or Factor VIII. Antibody binding was detected by incubation with secondary goat anti-mouse antibody conjugated to FITC and goat anti-rabbit conjugated with rhodamine. The upper panels show the co-localization of denatured collagen-IV and MMP-2 surrounding angiogenic blood vessels. The lower panels show the co-localization of denatured collagen-IV and factor VIII surrounding angiogenic blood vessels. Left panels indicate CAM tissue stimulated with bFGF. The right panels indicate CAM tissue with CS1 melanoma tumors growing in it. The red color in the upper panels indicates the expression of MMP-2 and in the lower panels indicates the expression of factor VIII. The green color in both the upper and lower panels indicates the expression of denatured collagen-IV. Yellow indicates co-location.

Figure 13 shows that Mab HUIV26 identifies denatured type IV collagen that surrounds blood vessels associated with human melanoma tumor. The generation and localization of denatured collagen IV in vivo was studied by indirect immunofluorescence in frozen tissue sections of human melanoma tumor biopsies. Frozen tissue sections of human melanomas were fixed in acetone, blocked with 1% BSA and co-stained with Mab HUIV26 and polyclonal antibody directed against factor VIII, a known marker of blood vessels. Antibody binding was detected by incubation with secondary goat anti-mouse antibody conjugated to FITC and goat anti-rabbit conjugated with rhodamine. Red indicates the expression of factor VIII and marks human blood vessels. Green indicates denatured collagen IV specifically associated with angiogenic blood vessels associated with tumor.

Figure 14 demonstrates the effects of Mab HUIV26 on the adhesion of human endothelial cell. Plates microtiter (96 wells) were coated with type IV collagen native or denatured. The wells of the plate microtiter were then blocked with 1% BSA in PBS for 1 hour at 37 ° C. Then, the cells were allowed human endothelials joined the coated wells in presence or absence of purified Mab HUIV26 or an antibody of control corresponding to isotype at a concentration of 100 / mug / ml for 30 minutes. Unbound cells were removed by washing and the bound cells were stained with crystal violet. TO then the cells were incubated with 10% acetic acid and the Cell adhesion was quantified by measuring optical density (D.O.) of dye eluted at 600 nm. Data bars represent the DO. mean ± standard deviation of wells in triplicate.

Figure 15 shows the effects of Mab HUIV26 on the migration of human endothelial cells. Membranes of transwell migration chambers were coated with collagen from native or denatured type IV. Then, the cells were allowed human endothelials will migrate to the lower side of the Coated membranes in the presence or absence of Mab HUIV26 purified or a control antibody corresponding to isotype (100  mug / ml) for a total of 6 hours. The cells that remained on the upper side of the membrane were removed and the cells that had migrated to the lower side of the membrane were stained with crystal violet Next, the membranes were incubated with 10% acetic acid and cell migration was quantified by measuring the optical density (D.O.) of dye eluted at 600 nm. The bars of data represent the D.O. mean ± standard deviation of triplicate wells.

Figure 16 demonstrates the effects of systematic administration of purified Mab HUIV26 on angiogenesis in vivo. Filter discs saturated with bFGF were placed on the Chorioallantoid Membranes (CAM) of 10-day-old chicken embryos. Twenty-four hours later, the embryos received a single intravenous injection with 20 µg of Mab HUIV26 or a control. At the end of a 3 day incubation period, the filter discs and surrounding CAM tissues were removed and the angiogenesis was quantified by counting the number of branching points of blood vessel in the area of the filter disc. Figure 6 shows examples of CAM tissue from a typical experiment.

Figure 17 shows the quantification of the angiogenesis experiments with the Mab HUIV26. Data bars represent the D.O. mean ± typical errors of 5 to 10 embryos by condition The angiogenesis index is equal to the number of branching points of experimentally treated embryos less the number of CAM branching points in the absence of bFGF.

Figure 18 shows the effects of systematic administration of purified Mab HUIV26 on tumor growth in vivo. CS-1 melanoma tumor cells (5 x 10 6) were inoculated into CAMs of 10-day-old chicken embryos. Twenty-four hours later, the embryos received a single intravenous injection of 20 µg of purified or control Mab HUIV26. The embryos were allowed to incubate for a total of 7 days. The photographs represent examples of embryonic tumors treated with control or HUIV26 within CAM tissue.

Figure 19 shows the size comparison of tumors removed. At the end of the incubation period of 7 days, the resulting tumors were removed and analyzed for size Global and wet weight. The photograph represents a comparison about the size of tumors removed from embryos treated with control or Mab HUIV26.

Figure 20 shows weight quantification in wet tumors. Data bars represent the average ± typical tumor weight errors of 5 to 10 embryos per condition.

Figure 21 shows the effects of Mab HUIV26 on tumor growth evaluated in a model system of SCID mouse tumor. SCID mice were injected subcutaneously 2 x 10 6 M21 human melanoma cells. Three days later it started a 24-day treatment with daily injections intraperitoneal 100 µg of Mab HUIV26, an antibody of control corresponding to isotype or without treatment. The volume of tumor was monitored by caliper measurements and the tumor volume Five mice were included in each group. The data represent the mean ± typical errors of the tumor volumes for each experimental condition. They were obtained similar results in two independent experiments in which five or ten mice were included in each condition experimental.

Figure 22 shows an analysis of the reactivity of Mab HUIV26 with collagen peptides containing GDPR Microtiter plates were coated with 50 µl of collagen peptides containing RGD (100 µg / ml) (A) or denatured human type IV collagen (B). The plates are blocked with 1% BSA in PBS to avoid non-specific binding. Mab HUIV26 (1.0 µg / ml, 50 µl / well) was allowed to cool. bind to the coated plate for 1 hour at 37 ° C. The union of Mab HUIV26 was detected by incubation with secondary antibody peroxidase labeled. Immunoreactivity was quantified by measuring optical density (D.O.) with a plate reader microtiter TO). Immunoreactivity of purified Mab HUIV26 against collagen peptides containing RGD. B. HUIV26 immunoreactivity that binds to type IV collagen denatured immobilized in the presence or absence of peptides of collagen containing soluble RGD or type IV collagen denatured. The data bars represent the D.O. half ± standard deviation of wells in triplicate.

Figure 23 shows cord formation Human endothelial on denatured type I collagen. Be coated Millipore flexible membranes with type I collagen native or denatured human. It was allowed endothelial cells human (HUVEC) will interact with collagen for 5 hours in absence of added growth factors or serum.

Figure 24 shows the analysis of the Endothelial cell survival on type I collagen. Microtiter wells were coated with collagen type 1 native or proteolized / denatured human. Then it let the HUVEC join the wells in the absence of factors of growth added or serum for 5 hours. At the end of the 5 hour incubation, bound cells were removed, fixed and stained for apoptosis with the ApopTag kit and analyzed by flow cytometry. Data indicated mean ± typical deviations of the percentage of human endothelial cells They were starting to experience apoptosis. The data is obtained from wells in triplicate.

Figure 25 shows the effects of domains of Type I cryptic collagen on cord formation endothelial Collagen I amino acid sequence analysis human and three-dimensional structure revealed potential cryptic domains that would be inaccessible within the Collagen I from triple mature helix. Peptides were generated synthetic corresponding to 5 of these cryptic domains potential human collagen I. These cryptic domains are immobilized in microtiter wells and performed endothelial cord formation assays as described previously. As shown, the 5 collagen peptides endured endothelial cell adhesion for 1 hour later of the sown. However, it was shown that the collagen peptide human 2 facilitates the formation of endothelial cord and enhances the endothelial cell survival after 18 hours while that the other peptides showed little, if any, activity at the time point of 18 hours.

Figure 26 shows that cryptic domains of type I collagen support endothelial cell adhesion by different integrin receptors. The peptides they represent cryptic domains of human collagen I were immobilized on microtiter wells. Endothelial cells were allowed to bind peptides in the presence or absence of antibodies to function block directed against specific integrins. Everybody the peptides supported cell adhesion at varying levels. The cell adhesion to the 5 peptides was dependent on the ligation of integrin? \ nu \ beta3 since the Mab LM609 directed against α? nu? 3 blocked cell adhesion. Surprisingly, cell adhesion to peptide 2 was also dependent on a β1 integrin, since cell adhesion to this peptide was also blocked by P4C10 directed against integrins β1.

Figure 27 demonstrates the Mab generation reagents with cryptic domains of type I collagen. Domains  Type I collagen cryptics were used to generate Mab. One of These Mab named XL313 was used for further study. Be immobilized human collagen I peptides over wells of microtiter and purified Mab XL313 binding was evaluated. How Shown below, the Mab XL313 specifically recognized the human collagen peptide 2. In addition, the Mab XL313 also recognized the collagen 4 peptide, however, the peptide of Collagen 4 is not present in mature collagen I. XL313 no reacted with other similar type I collagen peptides.

Figure 28 shows that Mab XL313 recognizes specifically denatured human type I collagen. Be coated microtiter plates with type I or collagen type IV native or denatured human. The capacity of Mab XL313 of binding to native or denatured human type I collagen and Denatured type IV collagen was tested by phase ELISA solid. Mab XL313 specifically recognized type collagen Denatured human I but not native type I collagen. In addition, Mab XL313 failed to bind to type IV collagen native or denatured human.

Figure 29 shows that Mab XL313 inhibits the chicken angiogenesis. Angiogenesis was induced in CAM of 10-day-old chicken embryos with bFGF. Twenty four hours later, the embryos received a single IV injection with 50 µg of the Mab XL313 or a control corresponding to isotype. Three days later, angiogenesis was quantified by counting the number of branching points of blood vessels within the area of the filter disc TO; representative examples of CAM tissue of a typical experiment

Figure 30 shows the quantification of the angiogenesis experiments of Figure 29. Data bars represent the mean ± typical errors of 5 to 10 embryos per condition.

Figure 31 shows the effects of the systemic administration of Mab XL313 on tumor growth of human fibrosarcoma. Human fibrosarcoma cells were inoculated HT1080 (5 x 10 6) in CAMs of 10-day chicken embryos old. Twenty-four hours later, the embryos received a single intravenous injection of Mab XL313 purified to 50 µg concentrations per embryo. After seven days, the tumors were removed and wet weights were determined. Quantification of tumor weight. Data bars represent the average ± typical errors of 5 to 10 embryos per condition.

Figure 32 shows that the mutation at the MMP cleavage site of collagen I inhibits the growth of melanoma tumor in vivo. Transgenic mice that house a mutation at the MMP cleavage site within collagen I were used to determine if type I collagen proteolysis can play a role in tumor growth and angiogenesis. Col al transgenic mice have a mutation that inhibits MMP binding and excision of type I collagen. B6 Col al transgenic mice or wild type control B6 mice were injected subcutaneously transgenic B 16 melanoma tumor cells The tumors were allowed to develop for 11 days and the tumor size was controlled with calipers. As shown below, B16 melanoma cells formed large proliferative tumors in mice that can easily proteolize type I collagen. In contrast, B16 melanoma cells showed little or no ability to form tumors in B6 Col transgenic mice. at which proteolysis of type I collagen is inhibited. Data represent the mean ± standard errors of tumor volumes of 5 mice per condition.

Figure 33 shows that Mab XL313 inhibits the tumor growth in B6 mice. Tumor growth of Lewis lung carcinoma was examined in B6 type mice wild or in transgenic mice Col al. TO; Were injected Lewis lung carcinoma cells subcutaneously in B6 wild type mice or B6 Col al transgenic mice Col. B; Lewis lung carcinoma tumor growth comparison in wild-type or transgenic B6 mice B6 Col al. C; TO wild-type B6 control mice were injected cells from Lewis lung carcinoma. Twenty four hours later, the mice were treated systemically with Mab XL313 or an antibody of control corresponding to isotype (100 µg / per injection). Be let the tumors develop for 11 days and the sizes of the tumor were controlled with calipers. The data represents the mean ± typical errors of tumor volumes of 5 mice by condition

Description of the invention Collagens

The composition of the invention is suitable for use with several collagen molecules, including those of any animal In one embodiment, the collagens are collagens. Humans. The collagens can also be of any mammal such like rat, mouse, pig, rabbit, etc. or of a bird such as a chicken. Generally, a collagen is an extracellular matrix protein that contains a sequence [Gly-Xaa-Xaa] n. The collagen types are well known in the art (see, for example, Olsen, B. R. (1995) Curr. Op. Cell. Biol. 5: 720-727; Kucharz, E. J. The Collagens: Biochemistry and Pathophysiology, Springer-Verlag, Berlin, 1992; Kunn, K. in Structure and Function of Collagen Types, eds. R. Mayne and R. E. Burgeson, Academic Press, Orlando). Human collagens They are the preferred collagens. Denatured collagen refers to collagen that has been treated in such a way that it no longer adopts predominantly the native triple helix shape. The Denaturation can be achieved by heating the collagen. In a embodiment, the collagen is denatured by heating during about 15 minutes at about 100 ° C. The denaturation can also be achieved by treating the collagen with a chaotropic agent. The appropriate chaotropic agents they include, for example, guanidinium salts. The denaturation of a collagen can be controlled, for example, by changes spectroscopic optical properties such as absorbance, circular dichroism or fluorescence of the protein, by resonance nuclear magnetic, by Raman spectroscopy or by any Another suitable technique. Denatured collagen refers to denatured full length collagens as well as to collagen fragments A collagen fragment can be any collagen sequence shorter than a collagen sequence native. For collagen fragments with substantially structure native, denaturation can be performed as for a native full length collagen. Fragments can also have a size so that they do not have a native structure significant or have regions without significant native structure of the native triple helix shape. Such fragments are completely or partially denatured without requiring the use of heat or a chaotropic agent. Collagen expression denatured includes proteolyzed collagen. Collagen Proteolized refers to a collagen that has been fragmented by the action of a proteolytic enzyme. In particular, the collagen proteolyzed can be prepared by treating the collagen with a metalloproteinase, such as MMP-1, MMP-2 or MMP-9, or treating the collagen with a cell extract that contains activity of degradation of collagen or is it that occurs naturally in the neovascularization sites in a tissue.

A cryptic epitope within a collagen is a sequence that is not exposed to recognition within a native collagen, but is able to be recognized by an antagonist of a denatured collagen. The sequence of cryptic epitopes can be identified by determining the specificity of a antagonist. Candidate cryptic epitopes can also be identify, for example, by examining the three-dimensional structure of a native triple helix collagen. Peptide sequences that are not exposed to solvent or that are only exposed partially solvent in the native structure are potential cryptic epitopes.

An epitope is the sequence or sequences of amino acids that are recognized by an antagonist of the invention. An epitope can be a linear peptide sequence or it can be composed of non-contiguous amino acid sequences. A antagonist can recognize one or more sequences, therefore, a epitope can define more than one amino acid sequence target different. Epitopes recognized by an antagonist can be determine by peptide mapping and sequence analysis techniques well known to the person skilled in the art.

Antagonists

The antagonists of the invention bind to a denatured collagen but bind substantially affinity reduced to the native form of collagen. An "affinity substantially reduced "is an affinity of approximately 3 times less than the affinity for denatured collagen, more preferably about 5 times smaller and even more preferably about 10 times smaller and even more preferably more than 10 times less. Also, "substantially minus "indicates a difference of at least about one 3 times difference when referring to relative affinities. The antagonists are preferably specific to any one of denatured type I, II, III, IV or V collagen and combinations thereof. In one embodiment, an antagonist is binds denatured type I collagen but binds affinity substantially reduced to native type I collagen and collagen Type II, III, IV and V denatured. In another embodiment, a antagonist binds to denatured type IV collagen but binds with substantially reduced affinity to type IV collagen native. In another embodiment, an antagonist binds to the collagens Type I, Type II, Type III, Type IV and Type V denatured but binds with substantially reduced affinity to type I, type II, type III, type IV and Type V natives.

Apparent affinities can be determined by methods such as immunoabsorbent assay linked to enzymes (ELISA) or any other familiar technique for specialist in the technique. True affinities can be measure using techniques known to the specialist in technique.

In one embodiment, the peptides containing epitopes recognized by an antagonist can be used on their own same. In one embodiment, the epitopes defined by the monoclonal antibodies HUI77, HUIV26 and XL313 by themselves will use as antiangiogenic compositions.

Union Essays

The invention also provides methods of assay to identify collagen candidate antagonists denatured for use in accordance with the present invention. In these test methods candidate antagonists are evaluated for determine your ability to bind to both denatured collagen as a native collagen and, in addition, can be evaluated to determine its potency in the inhibition of angiogenesis in a tissue.

ELISA

The first test measures the binding of antagonists to denatured or native collagen in the solid phase by ELISA. The test is useful with a variety of types of collagens, for example, the assay can be used with type I, II collagen, III, IV and V, as well as for other components of the matrix extracellular

The essay can also be used to identify compounds that show specificity for denatured forms but not native to collagen. The specificity test is performed processing ELISA in parallel in which you select an antagonist potential concurrently in separate test chambers for determine the ability to bind denatured collagen and native people.

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Denatured Collagen Antagonists they can also be identified by their ability to compete for the binding with an antagonist of the invention. For example, you can select alleged antagonists controlling their effect on the affinity of a known antagonist, such as HUI77, HUIV26 or XL313, in a binding assay, such as ELISA. Such antagonists they probably have the same specificity as HUI77 and recognize the same cryptic epitope. The selected antagonist assumptions by a scanning method of this type they can be attached to the collagen or antagonist. Antagonists can be selected from the alleged antagonists by conventional binding assays to determine those that bind to the collagen epitope denatured but not the known antagonist.

Antagonists can also be identified for its ability to join a solid matrix that contains a denatured collagen. Such alleged antagonists are collected after altering the conditions of the solution, such as concentration of salts, pH, temperature, etc. Assumptions antagonists are further identified by their ability to cross, under appropriate solution conditions, a solid matrix to which a native collagen has been fixed.

Angiogenesis Assays

The antagonists of the invention are also can test to determine their ability to modulate the angiogenesis in a tissue. Any suitable assay can be used known to the person skilled in the art to control such effects. Several such techniques are described in this document.

The second test measures chicken chorioallantoid membrane angiogenesis (CAM) and is called the CAM test. The CAM assay has been described in detail by others and has been used additionally to measure both angiogenesis and neovascularization of tumor tissues. See Ausprunk et al ., Am. J. Pathol., 79: 597-618 (1975) and Ossonski et al ., Cancer Res., 40: 2300-2309 (1980).

The CAM test is a well-recognized test model for angiogenesis in vivo because the neovascularization of whole tissue takes place and the chicken embryo blood vessels themselves grow in the CAM or in the tissue developed on the CAM.

As demonstrated in this document, the essay of CAM illustrates neovascularization inhibition based on both in the quantity as in the scope of growth of new vessels. In addition, it is easy to control the growth of any tissue transplanted in CAM, such as a tumor tissue. Finally the test is particularly useful because there is a control internal for toxicity in the test system. The embryo of chicken is exposed to any test reagent and therefore Embryo health is an indication of toxicity.

A third trial measures angiogenesis in the rabbit eye model in vivo and is called the rabbit eye test. The rabbit eye test has been described in detail by others and has been further used to measure both angiogenesis and neovascularization in the presence of angiogenic inhibitors such as thalidomide. See D'Amato et al . (1994) Proc. Natl Acad. Sci. 91: 4082-4085.

The rabbit eye test is a well-recognized test model for angiogenesis in vivo because the neovascularization process, illustrated by rabbit blood vessels that grow from the edge of the cornea into the cornea, is easily visualized. through the transparent cornea naturally of the eye.

Additionally, both scope and amount of stimulation or inhibition of neovascularization or neovascularization regression can be controlled so simple over time.

Finally, the rabbit is exposed to any test reagent and, therefore, rabbit health is a indication of the toxicity of the test reagent.

A fourth test measures angiogenesis in the mouse chimeric: human mouse model and is called the mouse chimeric assay. The assay has been described in detail by others and has been further described herein to measure the angiogenesis, neovascularization and regression of tumor tissues. See Yan, et al . (1993) J. Clin. Invest. 91: 986-996.

The chimeric mouse assay is a useful test model for angiogenesis in vivo because the transplanted skin grafts closely resemble histologically normal human skin and complete tissue neovascularization occurs where human blood vessels themselves grow. from grafted human skin to human tumor tissue on the surface of grafted human skin. The origin of neovascularization in the human graft can be demonstrated by immunohistochemical staining of the neovasculature with markers of human-specific endothelial cells.

The chimeric mouse assay demonstrates the neovascularization regression based on both the amount as in the extent of the growth regression of new vessels. In addition, it is easy to control the effects on the growth of any transplanted tissue on grafted skin, such as a tumor tissue Finally, the test is useful because there is an internal control for toxicity in the test system. The mouse chimeric is exposed to any test reagent and, therefore, Mouse health is an indication of toxicity.

Antibodies

The present invention describes, in a embodiment, denatured collagen antagonists in the form of antibodies that bind denatured collagen but that bind to native collagen with a substantially reduced affinity. The antibody antagonists can also inhibit the angiogenesis

The antibodies of the invention can be monoclonal or polyclonal. In one embodiment, the antibodies Used are monoclonal. A monoclonal antibody of this invention comprises antibody molecules that immunoreact with collagen denatured isolated, but that immunoreact with an affinity substantially reduced with the native form of collagen. In a embodiment, an antibody of the invention recognizes collagen of type I denatured with an affinity of at least approximately 3 times, more preferably at least about 5 times and much more preferably at least about 10 times more than the affinity for denatured type I collagen. A antibody of the invention may also preferably bind to type IV collagen denatured and binds affinity substantially reduced to native type IV collagen. The antibodies of the invention can also bind to each of the type I, II, III, IV and V collagen and bind to native forms of each collagen with substantially reduced affinity.

Preferred monoclonal antibodies that are preferably bind denatured collagen include antibodies  monoclonal having the immunoreaction characteristics of mAb HUI77, mAb HUIV26 or mAb XL313.

Antibody antagonists of the invention can be generated according to several methods known to those skilled in the art. For example, an animal can be immunized with a denatured collagen or a fragment thereof. Antibodies generated in this way can be selected both for their ability to bind denatured proteolyzed collagen and for an affinity substantially reduced by the native form of the same collagen. Antibodies can be generated, for example, by the "subtractive immunization" method (see, for example, Brooks, PC et al . (1993) J. Cell. Biol. 122: 1351-1359).

The expression "antibody or molecule of antibody "in the various grammatical forms is used in this document as a collective noun that refers to a population of immunoglobulin molecules and / or portions immunologically active immunoglobulin molecules, that is, molecules that contain a combination site with antibody or paratope

A "combination site with antibody" is the structural portion of an antibody molecule comprised by variable and hypervariable regions of heavy and light chain that specifically bind antigen.

Exemplary antibodies for use in the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule containing the paratope, including portions known in the art as Fab, Fab ', F (ab ') 2 and (F (v) and are also called antibody fragments.

In another preferred embodiment, the invention contemplates a truncated immunoglobulin molecule comprising a Fab fragment from a monoclonal antibody of this invention. The Fab fragment, which lacks an Fc receptor, is soluble and provides therapeutic advantages in serum half-life and Diagnostic advantages in the ways of using the Fab fragment soluble. The preparation of a soluble Fab fragment is known generally in the immunological technique and can be achieved through a variety of methods.

For example, portions (fragments) are prepared Fab and F (ab ') 2 antibody by reaction papain and pepsin proteolytic, respectively, in antibodies substantially intact by methods that are well known. See, for example, U.S. Patent No. 4,342,566 of Theofilopolous and Dixon. Portions of Fab 'antibodies also they know each other well and are produced from portions F (ab ') 2 followed by reduction of disulfide bonds which join the two heavy chain portions as with mercaptoethanol and followed by alkylation of the resulting mercaptan protein with a reagent such as iodoacetamide. An antibody is preferred which contains intact immunoglobulin molecules and is used as illustrative in this document.

The expression "monoclonal antibody" in its various grammatical forms refers to a population of molecules  of antibodies that contain only one kind of site of combination of antibody capable of immunoreacting with an epitope particular. A monoclonal antibody, therefore, may contain an antibody molecule that has a plurality of sites of combination of antibody, each immunospecific for an epitope  different, for example, a bispecific monoclonal antibody.

A monoclonal antibody is composed typically by antibodies produced by single cell clones  called a hybridoma that secretes (produces) only one type of antibody molecule. The hybridoma cell is formed by fusing an antibody producing cell and a myeloma cell line or another cell line with self-perpetuation. The preparation of such antibodies was first described by Kohler and Milstein, Nature 256: 495-497 (1975). Be describe additional methods by Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987). The supernatants of hybridoma prepared in this way can be screened for the presence of antibody molecules that immunoreact with denatured collagens.

In summary, to form the hybridoma from from which the monoclonal antibody composition is produced, a myeloma cell line or other cell line with self-perpetuation fuses with lymphocytes obtained from the spleen of a hyperimmunized mammal with a source of denatured collagen.

It is preferred that the myeloma cell line used to prepare a hybridoma of the same species as the lymphocytes Typically, a mouse of strain 129 GIX.sup. + Is the preferred mammal Mouse myelomas suitable for use in The present invention includes cell lines sensitive to hypoxanthine-aminopterin-thymidine (HAT) P3X63-Ag8.653 and Sp2 / 0-Ag14 that are available in the American Type Crop Collection, Rockville, Md., With the designations CRL 1580 and CRL 1581, respectively.

Splenocytes typically fuse with Myeloma cells using polyethylene glycol (PEG) 1500. Hybrids fused are selected for their sensitivity to a means of selective culture, such as HAT medium (hypoxanthine aminopterin thymidine) Hybridomas that produce a monoclonal antibody from This invention is identified using the immunoabsorbent assay Enzyme-linked (ELISA) described in the Examples.

A monoclonal antibody of the present invention can also be produced by initiating a culture of monoclonal hybridoma comprising a nutrient medium that It contains a hybridoma that secretes antibody molecules from the appropriate specificity. The crop is maintained in conditions and for a sufficient period of time for the hybridoma secrete the antibody molecules into the medium. The medium it contains antibody is then collected. The antibody molecules can be then further isolate by well known techniques.

Media useful for the preparation of these compositions are well known in the art and are commercially available and include synthetic culture medium, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimum essential medium (DMEM; Dulbecco et al ., Virol. 8: 396, 1959) supplemented with 4.5 g / l glucose, 20 nM glutamine and 20% fetal calf serum. An exemplary inbred mouse strain is Balb / c.

Other methods for producing a monoclonal antibody, a hybridoma cell or a hybridoma cell culture are also well known. See, for example, the method for isolating monoclonal antibodies from an immune repertoire as described by Sastry et al . (1989) Proc. Natl Acad. Sci. USA, 86: 5728-5732; and Huse et al . (1989) Science, 246: 1275-1281.

Also considered by this invention is the hybridoma cell and cultures containing hybridoma cells that produce monoclonal antibodies of this invention. It preferred particularly a hybridoma cell line that secretes the monoclonal antibody mAb HUI77, mAb HUIV26 or mAb XL313.

The invention considers, in one embodiment, a monoclonal antibody that has the characteristics of immunoreaction of mAb HUI77, mAb HUIV26 or mAb XL313.

It is also possible to determine, without excessive experimentation, if a monoclonal antibody has a equivalent specificity (immunoreaction characteristics) to a monoclonal antibody of this invention evaluating whether the first prevents the latter from joining a preselected target molecule. Yes the monoclonal antibody being tested competes with the monoclonal antibody of the invention, as shown by a decreased binding by the monoclonal antibody of the invention in conventional competition trials by binding to the target molecule when present in the solid phase, then it is likely that the two monoclonal antibodies bind to it epitope or one closely related.

An additional way to determine if a monoclonal antibody has the specificity of an antibody monoclonal of the invention is to determine the rest sequence amino acid of the CDR regions of the antibodies in question. Antibody molecules that have rest sequences identical or functionally equivalent amino acid in their CDR regions have the same binding specificity. In the technique methods for sequencing polypeptides are well known. This does not suggests that antibodies with different CDR regions cannot be join the same epitope.

The immunospecificity of an antibody, its binding ability to target molecule and related affinity that the antibody shown by the epitope are defined by the epitope with the one that immunoreacts the antibody. Epitope specificity is defined at least in part by the amino acid residue sequence of the variable region of the immunoglobulin heavy chain, the antibody and, in part, by the amino acid residue sequence of the variable region of the light chain.

The use of the expression "that has the binding specificity of "indicates that monoclonal antibodies equivalents show immunoreaction characteristics (binding) same or similar and compete for binding to a target epitope preselected

Humanized monoclonal antibodies offer particular advantages over monoclonal antibodies murine, particularly to the extent that they can be used Therapeutically in humans. Specifically, the antibodies humans don't clear up the circulation as quickly as "strange" antigens and do not activate its immune system so that strange antigens and strange antibodies. In the technique methods to prepare are generally well known "humanized" antibodies and can be applied easily  to the antibodies of the present invention.

Therefore, the invention considers, in a embodiment, a monoclonal antibody of this invention that is humanized grafting to introduce immune system components human without substantially interfering with the ability of the antigen binding antibody.

The antibody of the use of the invention can also be a fully human antibody such as those generated, for example, by selection of a phage display library of antibodies that have human single or double chain antibodies such as those described in Haard, HJ et al . (1999) J. Biol. Chem. 274: 18218-30 and in Winter, G. et al . (1994) Annu. Rev. Immunol. 12: 433-55.

Polypeptides

Denatured Collagen Antagonists they can also be polypeptides or peptides. The term polypeptide refers to a sequence of 3 or more amino acids connected between yes by peptide bonds between the amino alpha group and the group carboxy of contiguous amino acid residues. The term peptide as used in this document refers to a linear series of two or more connected to each other as in a polypeptide.

In one embodiment, the invention considers denatured collagen antagonists in the form of polypeptides. A denatured collagen polypeptide antagonist can be any peptide or polypeptide capable of binding to a collagen denatured, but binds to the native form of collagen with substantially reduced affinity.

The identification of antagonistic peptides of denatured preferred collagen having selectivity for denatured collagen can be easily identified in a typical inhibition of binding assay, such as in the ELISA assay described in the Examples.

Peptide antagonists can be generated and polypeptide by several techniques known to the specialist in the technique For example, a two-hybrid system (for example, Fields, S. (1989) Nature 340: 245-6) can use a fragment of a collagen as a "bait" to select protein antagonists of a library that binds to the peptide of collagen The library of potential antagonists can be produce from a cDNA library, for example. In other embodiment, potential antagonists may be variants of known collagen binding proteins. Such proteins can be randomize mutagenize or submit a combination of genes or other techniques available to generate sequence diversity.

Also antagonists of peptide and polypeptide by molecular evolution techniques. The Protein libraries can be generated by mutagenesis, combination of genes or other techniques available to generate molecular diversity The protein combinations they represent numerous variants can be selected for their ability to bind to denatured collagen, for example, by passing such protein combinations on a solid matrix that has been attached a denatured collagen. Elution with gradients of salt, for example, can provide purification of variants with affinity for denatured collagen. It can also influence a negative selection stage whereby such combinations are they pass on a solid matrix to which collagens have joined native people. The filtering will contain the variants within the combination that have a reduced affinity for the native form of collagen

Peptide and polypeptide antagonists can also be generated by phage display. A peptide or a randomized protein can be expressed on the surface of a phagemid particle as a fusion with a phage envelope protein. Monovalent phage display techniques are widely available (see, for example, Lowman HB et al . (1991) Biochemistry 30: 10832-8.). Randomized peptide or protein libraries expressing phages can be screened with a solid matrix to which a native collagen molecule has been attached. The remaining phage does not bind to native collagens or binds to native collagens with a substantially reduced affinity. Then, the phage is screened against a solid matrix to which a denatured collagen has been bound. The bound phage is isolated and separated from the solid matrix by a change in the solution conditions or, for a properly designed construction, by proteolytic cleavage of a linker region that connects the phage envelope protein with the peptide library or randomized protein. The isolated phage can be sequenced to determine the identity of the selected antagonist.

A polypeptide includes any analog, chemical fragment or derivative of a polypeptide whose sequence of amino acid residue is shown herein provided that the polypeptide is a denatured collagen antagonist, but not of native collagen. Therefore, a present polypeptide can be submit to various changes, substitutions, insertions and deletions when such changes provide certain advantages in its use. TO in this regard, a collagen antagonist polypeptide denatured of this invention corresponds to, instead of being identical to, the sequence of a peptide indicated when performed one or more changes and retains the ability to function as a denatured collagen antagonist in one or more of the assays as defined in this document.

Therefore, a polypeptide can be in any of a variety of forms of peptide derivatives, which include amides, protein conjugates, cycled peptides, polymerized peptides, analogs, fragments, chemically peptides modified and similar derivatives.

Other Antagonists

The antagonists can also be small organic molecules, such as natural products or compounds synthesized by conventional organic synthesis or organic combination synthesis. The compounds can be tested to determine your ability to bind to a collagen denatured, for example, by using the technique of column binding described above. The compounds they are also selected by reduced affinity for the native form of collagen by a similar column binding technique.

Antagonists can also be compounds. no peptides. Suitable non-peptide compounds include, by example, oligonucleotides. Oligonucleotides as used in this document refers to any heteropolymeric material that It contains purine, pyrimidine and other aromatic bases. The DNA and RNA oligonucleotides are suitable for use with the invention, such as oligonucleotides with modifications with sugar (for example, 2 'alkylated riboses) and main chain (for example, phosphorothioate oligonucleotides). Oligonucleotides they can present the bases found in common purine form and pyrimidine, such as adenine, thymine, guanine, citidine and uridine, as well as modified bases with the ring portion heterocyclic (for example, 7-desazaguanine) or in exocyclic positions. Oligonucleotide also includes heteropolymers with different structures that also have aromatic bases, which include polyamide nucleic acids and Similar.

An oligonucleotide antagonist can be generated by several methods known to the person skilled in the art. In one embodiment, a combination of oligonucleotides containing a large number of sequences is generated. The combinations can be generated, for example, by solid phase synthesis using monomer mixtures as an elongation step. The oligonucleotide combination is classified by passing a solution containing the combination on a solid matrix to which a denatured collagen or a fragment thereof has been fixed. The sequences within the combination that bind denatured collagen are retained in the solid matrix. These sequences are eluted with a solution of different saline concentration or pH. The selected sequences are subjected to a second selection stage. The selected combination is passed on a second solid matrix to which native collagen has been fixed. The column retains the sequences that bind the native collagen, thereby enriching the combination in specific sequences for the denatured collagen. The combination can be amplified and, if necessary, mutagenized and the process can be repeated until the combination shows the characteristics of an antagonist of the invention. Individual antagonists can be identified by sequencing members of the oligonucleotide combination, usually after cloning said sequences into a host organism such as E. coli .

Disease Treatment

The present invention relates in a manner general to the discovery that the ligation of certain epitopes in denatured collagen but no native collagen inhibits the angiogenesis This discovery is important due to the role that plays angiogenesis in a variety of pathological processes. Inhibiting angiogenesis can intervene in the disease, mitigate the symptoms and in some cases, cure the disease.

When the growth of new blood vessels is the cause of or contributes to the pathology associated with a disease, inhibition of angiogenesis will reduce the effects harmful of the disease. Examples include psoriasis, rheumatoid arthritis, diabetic retinopathy, diseases inflammatory, restenosis, macular degeneration and the like. When the growth of new blood vessels is required to support the growth of a harmful tissue, inhibition of angiogenesis will reduce the blood supply to the tissue and, by therefore, it will contribute to the reduction in tissue mass based on Blood supply requirements. Examples include the tumor growth where neovascularization is a requirement continued for the tumor to grow beyond a few millimeters thick and for the establishment of tumor metastases solid.

The use of the present invention is effective in part because the therapy is highly selective for angiogenesis and not for other biological processes. As shown In the Examples, only the growth of new vessels blood is inhibited by denatured collagen antagonists and, therefore, therapeutic methods do not affect Adverse to mature vessels. As also shown in the Examples, a antagonist binds to angiogenic sites in tumors but not to normal surrounding tissues.

The discovery that ligation or collagens denatured alone can effectively inhibit the angiogenesis allows the development of therapeutic compositions with potentially high specificity and therefore toxicity relatively low Therefore, although the invention describes the use of antibody-based antagonists that have the ability to bind one or more denatured collagens, others can be designed antagonists that can also specifically bind collagens denatured, but not native collagens.

Prior to the findings of the present invention, it was not known that angiogenesis and any of the angiogenesis-dependent processes could be inhibited in vivo by the use of reagents that antagonize cryptic epitopes in collagens, that is, those found in proteolyzed collagens or denatured, but not in native forms of the same collagens.

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Methods for Inhibition of Angiogenesis

The invention provides the use of a antagonist for the manufacture of a composition for the inhibition of angiogenesis in a tissue and, therefore, the inhibition of tissue events that depend on the angiogenesis Generally, the method comprises administering to tissue a composition comprising an inhibitory amount of angiogenesis of a denatured collagen antagonist.

As described above, the Angiogenesis includes a diversity of processes that involve the neovascularization of a tissue that includes "growth rapid ", vasculogenesis or augmentation of vessels, processes of the angiogenesis that all involve collagen alteration of extracellular matrix in blood vessels. With the exception of healing of traumatic wounds, corpus luteum formation and embryogenesis, it is thought that most of the processes of the angiogenesis are associated with pathological processes and, therefore therefore, the use of the present therapeutic methods are selective for the disease

There is a diversity of diseases in which angiogenesis is thought to be important, called angiogenic diseases, which include, but are not limited to, inflammatory disorders such as immune and non-immune inflammation, chronic joint rheumatism and psoriasis, disorders associated with inappropriate or inappropriate invasion of vessels such as retinopathy diabetic, neovascular glaucoma, restenosis, capillary proliferation in atherosclerotic plaques and osteoporosis and associated disorders with cancer, such as solid tumors, tumor metastases solids, angiofibromas, retrolental fibroplasia, hemangiomas, Kaposi's sarcoma and similar cancers that require neovascularization to support tumor growth. Others Suitable tumors include melanoma, carcinoma, sarcoma, fibrosarcoma, glioma and astrocytoma.

Therefore, methods that inhibit angiogenesis in a diseased tissue mitigate the symptoms of the disease and, depending on the disease, may contribute to the cure of the disease. In one embodiment, the invention considers the use of an antagonist for the manufacture of a composition for the inhibition of angiogenesis, per se , in a tissue. The extent of angiogenesis in a tissue and, therefore, the extent of inhibition achieved by the present methods, can be evaluated by a variety of methods, such as those described in the Examples for detecting immature vessel structures and immunopositive springs to proteolyzed or denatured collagen by immunohistochemistry.

As described in this document, anyone of a variety of tissues or organs comprised of tissues organized can withstand angiogenesis in pathological conditions which include skin, muscle, intestine, connective tissue, joints, bones and similar tissues in which the vessels Blood can invade after angiogenic stimuli. Fabric, as used in this document, also includes all body fluids, secretions and the like, such as serum, blood, cerebrospinal fluid, plasma, urine, synovial fluid, vitreous humor.

Therefore, a tissue to be treated is a tissue inflamed and angiogenesis to be inhibited is tissue angiogenesis inflamed when there is neovascularization of inflamed tissue. In this class, the method considers the inhibition of angiogenesis in arthritic tissues, such as in a patient with rheumatism chronic joint, in inflamed tissues immune or nonimmune, in psoriatic tissue and the like.

The patient desirably is a patient human, although it has to be understood that the principles indicate that the use of the invention is effective with respect to all mammals, which are intended to be included in the term "patient". In this context, it is understood that a mammal includes any species of mammal in which the treatment of diseases associated with angiogenesis, particularly species of agricultural and domestic mammals. Such patient can be, for example, a pig, a cow, a horse, a goat, a sheep, a mule, a donkey, a dog, a cat, a Rabbit, a mouse and a rat.

In another related embodiment, a tissue a treat is a retinal tissue of a patient with retinopathy diabetic, macular degeneration or neovascular glaucoma and angiogenesis to inhibit is angiogenesis of the retinal tissue where there is neovascularization of retinal tissue.

In a further related embodiment, a tissue to be treated is a tumor tissue of a patient with a tumor solid, a metastasis, a skin cancer, a breast cancer, a hemangioma or angiofibroma and similar cancer and angiogenesis to inhibit is angiogenesis of tumor tissue where it exists neovascularization of a tumor tissue. Tumor tissues typical solid that can be treated by the present methods include lung, pancreas, breast, colon, laryngeal, ovarian, sarcoma of Kaposi and similar tissues. Angiogenesis of tumor tissue Exemplary, and inhibition thereof, are described in the Examples

The use of an antagonist for the manufacture of a composition for tissue angiogenesis inhibition Tumor is a particularly preferred embodiment because of the important role that neovascularization plays in the tumor growth In the absence of tissue neovascularization tumor, the tumor tissue does not get the required nutrients, slows down growth, suspends further growth, returns  and finally it becomes necrotic resulting in the tumor destruction

In other words, the present invention provides a composition to inhibit neovascularization of the tumor by inhibiting the angiogenesis of the tumor or to inhibit the tumor growth

The methods are also particularly effective. against the formation of metastases because (1) their formation it requires vascularization of a primary tumor so that the metastatic cancer cells can leave the primary tumor and (2) its establishment in a secondary site requires neovascularization to support the growth of metastasis.

In a related embodiment, the invention consider the composition together with other therapies such as conventional chemotherapy directed against solid tumors and for the control of the establishment of metastases. The administration of Angiogenesis inhibitor is typically performed during or after of chemotherapy, although it is preferable to inhibit angiogenesis then a chemotherapy regimen at a time when the Tumor tissue will respond to toxic attack by inducing angiogenesis to recover the forecast of a blood supply and nutrients to the tumor tissue. In addition, it is preferred to administer the methods of inhibition of angiogenesis after surgery in the that solid tumors have been removed as a prophylaxis against metastasis.

To the extent that you present them compositions are applied to the inhibition of neovascularization of the tumor, the compositions can also be applied to the inhibition of tumor tissue growth, to inhibition of tumor metastasis formation and tumor regression established.

Restenosis is a migration process of smooth muscle cells (SMC) and proliferation at the site of percutaneous transluminal coronary angioplasty that hinders the Angioplasty success. Migration and proliferation of SMC associated with blood vessels during restenosis is related to the angiogenesis process that is inhibited by present methods. Therefore, the compositions also consider restenosis inhibition by inhibiting processes related to angiogenesis in a patient after angioplasty procedures For the inhibition of restenosis, the composition is typically administered after angioplasty procedure for about 2 to about 28 days, and more typically for about the first 14 days after the procedure.

Dosage intervals for denatured collagen antagonist depend on the shape of the antagonist and its potency, as further described in this document, and they are large enough quantities to produce the desired effect in which angiogenesis is mitigated and The symptoms of the disease mediated by angiogenesis. The Dosage should not be so large as to cause effects adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure and the like. Generally, the dosage will vary with age, condition, sex and extent of the disease in the patient and can be determined by The specialist in the art. The dosage can also be adjust by the individual physician in the event of any complication.

A therapeutically effective amount is an amount of denatured collagen antagonist sufficient to produce a measurable inhibition of angiogenesis in the tissue being treated, that is, an angiogenesis inhibitory amount. Angiogenesis inhibition can be measured in situ by immunohistochemistry, as described herein, or by other methods known to the person skilled in the art.

The potency of a denatured collagen antagonist can be measured by a variety of means including inhibition of angiogenesis in the CAM assay, in the rabbit eye test in vivo , in the mouse chimeric assay: human in vivo and similar essays

A therapeutically effective amount of a denatured collagen antagonist for use in the invention in the form of a monoclonal antibody is typically an amount such that when administered in a physiologically composition tolerable is enough to achieve a plasma concentration from about 0.01 microgram (µg) per milliliter (ml) to about 100 µg / ml, preferably about 1 µg / ml at about 5 µg / ml and usually approximately 5 µg / ml. In other words, the dosage it can vary from about 0.1 mg / kg to about 300 mg / kg, preferably about 0.2 mg / kg a about 200 mg / kg, more preferably about 0.5 mg / kg to approximately 20 mg / kg, in one or more administrations of doses daily, for one or several days.

When the antagonist is in the form of a fragment of a monoclonal antibody, the amount can be adjusted in a simple way based on the mass of the fragment with respect to the mass of the complete antibody. A plasma concentration preferred in molarity is antibody antagonist of about 2 micromolar (µM) to about 5 millimolar (mM) and preferably about 100 µM to 1 mM.

A therapeutically effective amount of a denatured collagen antagonist for the use of the invention in the form of a polypeptide, or small molecule, it is typically a amount of polypeptide such that when administered in a physiologically tolerable composition is enough to get a plasma concentration of approximately 0.1 microgram (mug) per milliliter (ml) at approximately 200 mug / ml, preferably from about 1 µg / ml to about 150 \ mug / ml. Based on a polypeptide that has a mass of approximately 500 grams per mole, plasma concentration preferred in molarity is polypeptide antagonist of about 2 micromolar (µM) to about 5 millimolar (mM) and preferably about 100 µM to 1 mM. Saying otherwise, the dosage per body weight may vary from about 0.1 mg / kg to about 300 mg / kg and preferably from about 0.2 mg / kg to about 200 mg / kg, in one or more dose administrations daily, during One or several days.

Monoclonal antibodies or polypeptides for The use of the composition of the invention can be administered by parenteral route by injection or by gradual infusion along weather. Although the tissue to be treated may be accessible typically in the body by systemic administration and, therefore therefore, it can be treated frequently by administration intravenous therapeutic compositions, are considered other tissues and means of supply when there is a probability of that the target tissue contains the target molecule. By therefore, antagonists that include monoclonal antibodies, polypeptides and derivatives thereof can be administered by intravenously, intraperitoneally, intramuscularly, by subcutaneous route, intracavitary route, transdermal route, by topically, intraocularly, orally, intranasally, and They can be supplied by peristaltic means.

Therapeutic compositions containing a monoclonal antibody or a polypeptide of this invention is conventionally administered intravenously, as per injection of a unit dose, for example. The expression "dose unit "when used with reference to a composition Therapeutic of the present invention refers to units physically discrete suitable as unit dosage for the subject, each unit containing a predetermined amount of active material calculated to produce the therapeutic effect desired in association with the required diluent; that is to say, excipients or vehicle.

In one embodiment as shown in the Examples, the denatured collagen antagonist is administered. in a single intravenous dosage.

The compositions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered and the timing depend on the patient to be treated, the ability of the patient's system to use the active ingredient and the degree of therapeutic effect desired. The precise amounts of active ingredient required to administer depend on the discretion of the physician and are peculiar to each individual. However, suitable dosage ranges for systemic application are described in this document and depend on the route of administration. Suitable regimens for administration are also variable, but are typified by an initial administration followed by repeated doses at intervals of one or more hours by a subsequent injection or other administration. Alternatively, a continuous intravenous infusion is considered sufficient to maintain blood concentrations at the intervals specified for in vivo therapies .

As demonstrated by the present Examples, the inhibition of angiogenesis and tumor regression take place as early as 7 days after initial contact with the antagonist. Additional or prolonged exposure to antagonist is preferable for 7 days to 6 weeks, preferably about 14 to 28 days.

Therapeutic Compositions

The present invention considers compositions therapeutic Therapeutic compositions herein invention contain a physiologically tolerable vehicle together with a denatured or proteolyzed collagen antagonist as described herein, dissolved or dispersed therein as An active ingredient. The collagen antagonist composition denatured therapeutic is not immunogenic when administered to a mammalian or human patient for therapeutic purposes.

As used in this document, the expressions "pharmaceutically acceptable", "physiologically tolerable "and grammatical variations thereof, when refer to compositions, vehicles, diluents and reagents, are use interchangeably and represent that the materials are capable of administration to or on a mammal.

The preparation of a pharmacological composition which contains active ingredients dissolved or dispersed in the it is well understood in the art and does not have to be limited based on the formulation. Typically, such compositions are prepare as injectables, solutions or liquid suspensions, without However, solid forms suitable for solution, or suspensions, in liquid before use. The preparation It can also be emulsified.

The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in therapeutic methods described in this document. Excipients suitable are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. Further, if desired, the composition may contain smaller amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffering agents and the like that improve the effectiveness of the active ingredient.

The therapeutic composition of the present invention may include pharmaceutically acceptable salts of the components in this document. The salts pharmaceutically Acceptable include acid addition salts (formed with free amino groups of the polypeptide) that are formed with acids inorganic such as, for example, hydrochloric acid or phosphoric, or organic acids such as acetic, tartaric, Mandelic and the like. The salts formed with the carboxyl groups free can also be obtained from inorganic bases such as, for example, sodium hydroxide, potassium, ammonium, calcium or ferric and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, Procaine and the like. TFA salts are particularly preferred and HCl.

Physiologically tolerable vehicles are They know well in the art. They are copies of liquid vehicles sterile aqueous solutions that do not contain materials in addition of the active ingredients and water or containing such a buffer as sodium phosphate at physiological pH value, saline solution physiological or both, such as saline buffered with phosphate. Still additionally, aqueous vehicles may contain more than one buffer salt, as well as salts such as sodium chloride and potassium, dextrose, polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition and for the exclusion of water. They are copies of such additional liquid phases glycerin, oils vegetables such as cottonseed oil and emulsions of water oil.

A therapeutic composition contains a angiogenesis inhibitory amount of a collagen antagonist  denatured of the present invention, typically formulated to contain an amount of at least 0.1 percent by weight of antagonist by weight of total therapeutic composition. A percentage by weight is a weight ratio of inhibitor to total composition. Therefore, for example, 0.1 percent by weight is 0.1 grams of inhibitor per 100 grams of total composition.

An antibody can be conjugated with cytotoxins, cytotoxic agents, for delivery to a tumor or other tissue undergoing angiogenesis. Such conjugates can be prepared with a cytolysin or a hexotoxin, for example, ricin A, diphtheria toxin A or Pseudomonas exotoxin and fragments thereof. The cytotoxic agent can also be radiolabelled with an isotope in order to locally deliver a toxic dose of radioactivity to an angiogenic tissue.

The antagonists of the invention are also they can use to deliver an enzyme to a target in which the enzyme is able to convert a prodrug into an active form of the drug. Enzyme activated prodrug therapy directed at antibody (ADEPT) (see, for example, Syrigos, K. N. (1999) Anticancer Res. 19: 605-13). In short, a antagonist of the invention is conjugated to an enzyme, such as a lactamase, protease or esterase, which can convert a prodrug non-toxic or inactive in a toxic or active drug. Because the antagonist of the invention is located at angiogenesis sites and, particularly, at sites of tumors or metastases, the drugs Toxic can be directed to

Detection Methods

The antagonists of the invention are also suitable for use for the preparation of a composition of diagnosis for the detection of angiogenesis in tissues.

For example, when the antagonist is an antibody, the antagonist can be used in immunohistochemical techniques to stain ex vivo tissues. Immunological techniques such as immunostaining and ELISA are described, for example, in Binding Techniques Receptor, Methods in Molecular Biology, 106. ed. M. Keen. Humana Press, 1999; Brooks et al . (1998) Cell 92: 391-400; Brooks et al . (1996) Cell 85: 683-693; and Brooks et al . (1993) J. Cell. Biol. 122: 1351-1359.

The antagonist used in the invention, once attached to the target tissue, it can be detected directly or indirectly. Direct detection can be performed in antagonists comprising a detectable marker such as a fluorochrome, a radioactive marker, a paramagnetic heavy metal or a diagnostic dye.

Alternatively, the detection can take place through a secondary interaction. For example, an antibody detectably labeled that recognizes the antagonist can use to visualize the location of the antagonist. For example, if the antagonist is a monoclonal antibody of mouse origin, it you can use a goat anti-mouse antibody that is properly marked. The examples describe the use, by example of a goat anti-mouse antibody peroxidase conjugate. The specialist in the art can determine appropriate secondary antibodies for use with various antagonists

For in vivo detection, it is preferable to use a detectably labeled antagonist. The labeled antagonist is administered to a patient intravenously, intramuscularly, etc. Markers suitable for detection in a patient are particularly preferred. For example, paramagnetically labeled ones can be detected by magnetic resonance imaging. Radioactively labeled antagonists can also be detected.

Examples Example 1 HUI77 monoclonal antibody

This example describes the generation of a monoclonal antibody specific for denatured collagen, mAb HUI77.

The HUI77 mAb was generated and isolated by Immunological technique called subtractive immunization (S.I). The subtractive immunization technique allows to manipulate experimentally the immune response in mice to improve selectively an immune response to a rare epitope and / or not abundant in a mixture of common epitopes highly antigenic In summary, BALB / c female mice were injected via intraperitoneal collagen type I or type IV triple helix native human 24 and 48 hours after collagen injections triple helix, the mice were injected with the tolerizing agent cyclophosphamide to destroy activated B cells that would produce antibodies directed against common immunodominant epitopes in Type I and Type IV native triple helix collagen. After of the tolerization protocol, then the mice are injected denatured human type I or type IV collagen thermally to stimulate an immune response to epitopes exposed after thermal denaturation. Collagen is denatured by boiling for 15 minutes. Injections of type I and type IV thermally denatured collagen will administered every three weeks for a total of 4 to 5 injections The sera of each mouse were tested for immunoreactivity with both native triple helix collagen and denatured The mice that demonstrated the greatest title for reactivity against denatured collagen compared to the triple helix collagen were used for the production of hybridomas Spleen cells of the mice were fused selected with myeloma cells by techniques conventional. Individual hybridoma clones were tested. for the production of antibody against type I and type collagen IV triple helix or denatured. The clones were selected of hybridoma that produced antibodies that demonstrated a selective reactivity against type I or type IV collagen denatured compared to type I and type IV collagen of native triple helix. The Mab were purified by techniques conventional.

As shown in Figure 1, it was shown that HUI77 specifically recognizes type I and type IV collagen denatured but binds to type I and type IV collagens of native triple helix with substantially reduced affinity. In In particular, HUI77 binds to type I collagen denatured with an apparent reactivity of at least about 10 times superior to that of native type I collagen as measured by ELISA. HUI77 also binds denatured type IV collagen with a affinity of approximately 10 times higher than that of collagen of native type IV. In addition, the HUI77 mAb does not bind substantially to other matrix components, such as laminin, fibronectin, vitronectin or fibrinogen, thus demonstrating its specificity by a cryptic epitope within type I and type collagen IV.

The HUI77 mAb is also specific for others denatured collagen and binds to the native forms of these collagens with substantially reduced affinity. As it is shown in Figure 2, HUI77 also binds to collagen III, IV and V denatured approximately 7 times, approximately 8 times and approximately 10 times more closely than native forms respective of these collagens using ELISA.

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Example 2 Solid Tumor Detection

This example shows that antagonists of the invention can be used to detect denatured collagen in tumor tissue. HUI77 monoclonal antibody, described in Example 1, was used to indirectly immunostain normal and tumor tissue. As shown in Figure 3, indirect immunofluorescence analysis using the HUI77 mAb of human melanoma tumor biopsies as well as M21 melanoma tumor developed in full-thickness human skin indicates the generation of denatured forms of collagen associated with melanoma tumors. Human in vivo . To a large extent, little or no evidence of denatured collagen was detected in normal tissues in the absence of tumors, suggesting that denatured collagen may be a specific marker of solid human tumors.

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Example 3 Detection of Angiogenesis in Human Tumors

This example demonstrates that antagonists of the invention can be used to detect angiogenesis in human tumors. The indirect immunofluorescence analysis, shown in Figure 4, of human melanoma tumor biopsies demonstrates the generation and localization of denatured collagen surrounding blood vessels associated with human tumor in vivo. To a large extent, little or no evidence of denatured collagen was detected surrounding the HUI77 mAb normal blood vessels in the absence of tumors, suggesting that denatured collagen may be a specific marker of blood vessels associated with angiogenic tumor.

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Example 4 Antagonists that Inhibit Cell Adhesion and Migration Endothelial

This example demonstrates that certain antagonists of the invention can inhibit adhesion of endothelial cells human to denatured collagens.

The HUI77 mAb showed the ability to inhibit the adhesion of human endothelial cells to approximately 40% denatured type I collagen compared to the control antibody. These findings, summarized in Figure 5, suggest that the HUI77 mAb binds to a cryptic epitope in type I collagen that is at least partially involved in the adhesion of endothelial cells to denatured collagen I. Since endothelial cell adhesion processes are thought to play a role in tumor growth and angiogenesis, this function blocking antibody can have an effect on angiogenesis and tumor growth.
in vivo

The HUI77 mAb also showed the ability to inhibit the migration of human endothelial cells in approximately 80% denatured collagen compared to control antibody or no treatment, as shown in Figure 6. These findings suggest that the HUI77 mAb binds to a cryptic epitope in type I collagen that plays a significant role in cell migration in denatured collagen I. Since cell migration is thought to play an important role in tumor metastasis and angiogenesis and that denatured collagen is detected in association with malignant tumor cells and angiogenic blood vessels, this function blocking antibody can have a significant impact. about angiogenesis and tumor growth and metastasis in vivo.

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Example 6 Inhibition of Angiogenesis by the Monoclonal Antibody HUI77

This example shows that the antagonists of the invention effectively inhibit angiogenesis in the assay of Chicken CAM

In addition, systemic administration of mAb HUI77 inhibited βFGF-induced angiogenesis approximately 90% compared to controls (Figures 7 and 8). The angiogenic index was measured by counting the number of points of blood vessel branching in the chicken CAM test (Figure 8). To a large extent, no toxic side effects were observed in the embryos during the trial period. In addition, they were observed few or no effects of this Mab on quiescent blood vessels normal. It is possible that a concentration can be used a lot less than Mab and result in similar effects. These findings indicate that Mab HUI77 is a reagent potent anti-angiogenic that you can have significant clinical applications.

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Example 7 Inhibition of Tumor Growth by Mab HUI77

This example shows that antagonists of the invention effectively inhibit tumor growth in melanoma tumors in vivo.

Systemic administration of mAb HUI77 inhibited melanoma tumor growth approximately 53% compared to controls, as shown in Figures 9 and 10. To a large extent, no toxic side effects were observed in the embryos during the trial period. In addition, they were observed few or no effects of this Mab on adjacent tissue. Is it is possible that much lower concentrations of Mab can be used and result in similar effects. These findings indicate that the Mab HUI77 is a powerful anti-tumor reagent that It can have significant clinical applications.

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Example 8 HUIV26 Monoclonal Antibody

The Mab HUIV26 was generated using the technique immunological called subtractive immunization (S.I.) as has been indicated in Example I. As shown in Figure 11, it was shown that HUIV26 specifically recognizes type IV collagen denatured but does not bind to type I or type IV collagen of native triple helix. In addition, the Mab HUIV26 does not join others matrix components such as Laminin, Fibronectin, Vitronectin or fibrinogen, thus demonstrating its specificity by a cryptic epitope within type IV collagen.

As shown in Figure 11B, on site or cryptic sites in type IV collagen recognized by Mab HUIV26 become apparent after exposure to medium conditioning for HUVEC. The amount of reactivity with the Mab HUIV26 increased over the 24-hour period examined. This indicates that these endothelial cells secrete a capable protease of unmasking the cryptic site in type IV collagen recognized by the Mab HUIV26. The protease produced by HUVEC that unmask the cryptic site in type IV collagen is inhibited by the chelator EDTA (Figure 11B). This suggests that a Metalloprotease is responsible for initiating the unmasking of site or cryptic sites in type IV collagen. As shown  in Figure 12, the metalloprotease, MMP-2, is colocalize with the site or cryptic sites in collagen type IV that reacts with Mab HUIV26 at angiogenic sites in the CAM fabric. The serine protease inhibitor, aprotinin, has little effect on the unmasking of the site or sites Type IV collagen cryptics after one hour incubation with air conditioning for HUVEC (Figure 11B). In the points of time of 6 and 24 hours, the presence of aprotinin blocked 40 and 70 percent respectively of the reactivity observed in absence of protease inhibitor. This suggests that in the points later time (6 and 24 hours), serine proteases contribute to the unmasking of the site or cryptic sites in type IV collagen.

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Example 9 Arrest of Angiogenesis and Tumors with HUIV26

This example shows that you can use a antagonist to detect angiogenic processes in tissues.

As shown in Figure 12, indirect immunofluorescence analysis of chicken CAM tissue indicates the generation and localization of the denatured type IV collagen associated with angiogenic blood vessels induced by? FGF or tumor in vivo . To a large extent, little or no evidence of denatured collagen IV was detected in normal CAM tissues in the absence of bFGF or tumors, suggesting that denatured collagen IV may be a specific marker of angiogenic blood vessels in vivo.

Indirect immunofluorescence analysis, shown in Figure 13, of human melanoma tumor biopsies demonstrates the generation and localization of denatured collagen IV surrounding blood vessels associated with human tumor in vivo . To a large extent, little or no evidence of denatured collagen IV surrounding normal blood vessels was detected in the absence of tumors, suggesting that denatured collagen may be a specific marker of blood vessels associated with angiogenic tumor.

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Example 10 Inhibition of Migration and Cell Adhesion by Mab HUIV26

This example shows that a Mab antagonist It can inhibit the migration and adhesion of endothelial cells.

Mab HUIV26 showed the ability to inhibit the adhesion of human endothelial cells to approximately 70% denatured collagen IV compared to the control antibody (Figure 14). These findings suggest that Mab HUIV26 binds to a cryptic epitope in type IV collagen that is at least partially involved in the adhesion of endothelial cells to denatured collagen IV. Given the tissue distribution of type IV collagen and the fact that cell adhesion processes are thought to play a role in tumor growth and angiogenesis, this function blocking antibody can have a significant effect on angiogenesis and tumor growth in vivo.

Mab HUIV26 showed the ability to inhibit the migration of human endothelial cells in denatured collagen IV approximately 70% compared to the control antibody or no treatment. These findings suggest that Mab HUIV26 binds to a cryptic epitope in type IV collagen that plays a significant role in cell migration in denatured collagen IV. Since cell migration is thought to play an important role in tumor metastasis and angiogenesis and that denatured collagen was detected in association with angiogenic blood vessels, this function blocking antibody can have a significant impact on angiogenesis, growth of tumor and metastasis in vivo.

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Example 11 Inhibition of Angiogenesis by Systemic Administration of HUIV26

Systemic administration of Mab HUIV26 inhibited approximately 90% bFGF-induced angiogenesis in comparison with controls, as shown in Figures 16 and 17. To a large extent, no toxic side effects were observed in the embryos during the trial period. In addition, they were observed few or no effects of this Mab on blood vessels normal quiescent It is possible that a concentration can be used  much lower than Mab and result in similar effects. These findings indicate that Mab HUIV26 is a reagent potent anti-angiogenic that you can have significant clinical applications.

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Example 12 Tumor Growth Inhibition

Systemic administration of Mab HUIV26 inhibited the growth of melanoma tumor approximately 80% compared to controls (Figure 18). To a large extent, I don't know observed toxic side effects on embryos during trial period In addition, few or no effects of this Mab on adjacent tissue. They are currently being performed additional experiments to determine an IC50 value since it is it is possible that a much lower concentration of Mab can be used and given As a result similar effects. These findings indicate that the Mab HUIV26 is a powerful anti-tumor reagent and that It can have significant clinical applications.

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Systemic administration of Mab HUIV26 inhibited the growth of melanoma tumor in SCID mice. After  of 24 days of treatment, mice treated with Mab HCTIV26 had an average tumor volume less than 5 percent at observed in mice treated with a control Mab or in mice that They do not receive treatment.

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Example 13 Specificity of HUIV26 Epitope

This example shows that HUIV26 does not join RGD sequences in denatured collagens.

As shown in Figure 22, the Mab HUIV26 is not able to react with any of the peptides that contain RGD (arginine-glycine-aspartic acid) immobilized found in type IV collagen (Table 1). Six peptides that contain different soluble RGD found in non-collagen they managed to block the recognition of type IV collagen denatured immobilized by the Mab HUIV26 (Figure 22). These data suggest that Mab HUIV26 does not recognize GDPR sequences found in type IV collagen.

TABLE 1 RGD Domains of Type IV Collagen Human

one

Example 14 XL313 Monoclonal Antibody

Mab XL313 was generated by immunization with a synthetic peptide, whose sequence was obtained from type I collagen human. The sequence was chosen because it is included within the three-dimensional structure of type I collagen. The sequence of the synthetic peptide of 11 amino acid residues used was:

SEQ ID NO. 12: CysGlnGlyProArgGlyAspLysGlyGluCys

The KGE sequence (LysGlyGlu) was observed to be Very important for the recognition of XL313. The Mab XL313 joins specifically to the peptides of SEQ ID No. 1, but it has a binding affinity substantially decreased by peptides in the that the KGE sequence has been mutated:

SEQ ID NO. 13: CysGlnGlyProArgGlyAspAlaAlaAlaCys

Mab called XL313 is an antibody highly specific that reacts with type I collagen Proteolized / denatured. To a large extent, the XL313 does not react with the triple helix form native to type I collagen.  shown in Figure 27, Mab XL313 recognizes a cryptic domain of human collagen I, but not other similar peptides. These dates suggest that Mab XL313 may be a useful reagent to evaluate the role of the cryptic collagen domain defined by the peptide of Human collagen 2 in angiogenesis and tumor growth. How shown in Figure 28, Mab XL313 specifically recognizes a cryptic domain in human collagen I that is not exposed in the Mature triple helix conformation. In addition, this cryptic domain seems to be specific for collagen I since XL313 does not present cross-reactivity with native triple helix collagen IV.

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Example 15 Mab XL313 Inhibits Cell Adhesion and Migration

As shown in Figure 23, at the end of 5 hours of incubation, the HUVEC that had been allowed to bind to collagen I native formed a confluent monolayer. On the contrary, the HUVEC bound to denatured collagen began to migrate and to morphologically rearrange to form structures similar to cord. These data suggest that the hidden cryptic domains in the three-dimensional structure of human collagen I, which is not they can evaluate in their native triple helix state, they can play a role in morphogenesis and cord formation endothelial

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Example 16 Denatured Collagen Suppresses Apoptosis

As shown in Figure 24, the human endothelial cell interactions with collagen proteolyzed suppress apoptosis compared to collagen I native or endothelial cells maintained in suspension. These dates suggest that the cryptic domains hidden in the structure three-dimensional human collagen I, which cannot be evaluated in its native triple helix state, can play a role in the endothelial cell survival.

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Example 17 The Epitope Recognized by XL313

As shown in Figure 25, the human collagen cryptic peptide 2 appears to support endothelial cell survival and cord formation, while similar cryptic peptides present in human collagen I show little or no effect. These data suggest that the cryptic region of collagen I defined by peptide 2 may play an important role in angiogenesis and tumor growth in vivo.

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Example 18 Integrin Paper

As shown in Figure 26, integrin ? \ mu \ beta3 seems to play a leading role in the mediation of cellular interactions with all domains of cryptic collagen peptide I tested. Interestingly, the peptide 2 was also dependent on integrin interaction β1. These data suggest that peptide 2 supports cellular interactions by 2 different integrins.

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Example 19 Mab XL313 Inhibits Angiogenesis and Growth of Tumor

As shown in Figures 29 and 30, the Systemic administration of Mab XL313 inhibited angiogenesis the Chicken CAM model at more than 95% compared to controls. These data suggest that the cryptic domain of collagen I defined by the Mab XL313 plays an important role in the angiogenesis

As shown in Figure 31, Mab XL313 potentially inhibits the growth of HT1080 fibrosarcoma tumor in vivo . These findings indicate that the cryptic domain defined by Mab XL313 can play a significant role in the regulation of tumor growth in vivo.

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Example 20 Collagen Proteolysis is Important for the Growth of Tumor

As shown in Figure 32, in this experiment transgenic mice were used in which a mutation was introduced at the MMP cleavage site of the collagen I molecule, since those mice have an altered ability to proteolize their collagen I. To a large extent, B16 melanoma cells showed little or no ability to form tumors in mice that show altered collagen I proteolysis compared to wild-type control mice. These data suggest that collagen proteolysis I plays an important role in tumor growth in vivo.

As demonstrated with B16 melanoma cells, Lewis lung carcinoma cells showed little or no ability to form tumors when injected into B6 Col transgenic mice that had altered ability to proteolize their collagen I, while carcinoma cells Lewis lungs form rapidly growing tumors in control B6 mice (Figure 33). To a large extent, Mab XL313, specifically targeting a cryptic domain in collagen I that is exposed only after proteolysis, inhibited the growth of Lewis lung carcinoma tumor in approximately 80% wild-type B6 mice. The findings suggest that proteolytic exposure of the cryptic domain of collagen I in vivo may play an important role in tumor growth. In addition, these data suggest that Mab XL313 is a specific inhibitor of angiogenesis and tumor growth in vivo.

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<211> 11

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<212> PRT

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<213> Artificial Sequence

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         \ vskip0.400000 \ baselineskip
      

<220>

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<223> Description of Artificial Sequence: Sequence ID No. 12 in which the sequence has been mutated (LysGlyGlu)

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         \ vskip0.400000 \ baselineskip
      

<400> 13

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         \ hskip1cm
      

fifteen

Claims (30)

1. Use of an antagonist that binds specifically to a collagen or denatured collagen but it joins the triple helix form native to each of said collagen or collagens with substantially reduced affinity, in the that said antagonist inhibits angiogenesis, in which said antagonist is an antibody for the manufacture of a composition for the inhibition of angiogenesis in tissue induced by inflammation, psoriasis, macular degeneration or restenosis or during tumor growth or metastasis. 2. The use according to claim 1, in which said reduced affinity is approximately 3 times less than that of said denatured collagen, preferably approximately 5 times less than that for said collagen denatured, more preferably about 10 times less than for said denatured collagen. 3. The use according to claim 1 or 2, wherein said denatured collagen is type I collagen denatured, type II collagen denatured, collagen denatured type III, denatured type IV collagen or denatured type V collagen. 4. Use in accordance with any one of the claims 1 to 3, wherein said antagonist is an antibody  which has the binding specificity of the monoclonal antibody produced by the HUI77 hybridoma (ATTC Accession Number PTA-6551), HUIV26 (ATTC Access Number PTA-6563) or XL313 (ATTC Access Number PTA-6552). 5. The use according to claim 4, in which said antibody is a humanized monoclonal antibody or chemically modified 6. Use in accordance with any one of the claims 4 or 5, wherein said antibody is a fragment of a monoclonal antibody. 7. Use in accordance with any one of the claims 4 to 6, wherein said antagonist is conjugated with cytotoxic or cytostatic agents. 8. Use in accordance with any one of the claims 1 to 7, wherein said composition is administered intravenously, transdermally, intra-synovially, intramuscularly, intratumorally, intraocularly, intranasally, intrathecally, topically, via oral, in conjunction with chemotherapy or together with radiation. 9. Use in accordance with any one of the claims 1 to 8, wherein the tissue is present in a mammal, especially in which the tissue is arthritic, ocular, retinal or hemangioma. 10. Use in accordance with any one of the claims 1 to 9, wherein the tumor or metastasis is a melanoma, carcinoma, sarcoma, fibrosarcoma, glioma or astrocytoma 11. An antagonist, in which said antagonist is an antibody that specifically binds to a collagen or denatured collagen but that binds to the triple form native helix of each of said collagen or collagens with substantially reduced affinity, wherein said antagonist inhibits angiogenesis and is an antibody that has the binding specificity of monoclonal antibody produced by the HUI77 hybridoma (ATTC Accession Number PTA-6551), HUIV26 (ATTC Access Number PTA-6563) or XL313 (ATTC Access Number PTA-6552). 12. The antagonist of claim 11, in which said antibody is a humanized monoclonal antibody or chemically modified 13. The antagonist according to any one of claims 11 or 12, wherein said antibody is a fragment of a monoclonal antibody. 14. The antagonist according to any one of claims 11 to 13, wherein said antagonist is conjugates with cytotoxic or cytostatic agents. 15. Use of an antagonist that binds specifically to a collagen or denatured collagen but that joins the triple helix form native to each of said collagens or collagens with substantially reduced affinity, in the that said antagonist inhibits angiogenesis and in which said antagonist is an antibody for the preparation of a composition diagnostic, where said composition is able to detect angiogenesis, tumors or tumor inversion in a tissue putting in contact the composition with said tissue. 16. The use according to claim 15, wherein said reduced affinity is approximately 3 times lower than for said denatured collagen, preferably approximately 5 times less than that for said collagen denatured, more preferably about 10 times less than for said denatured collagen. 17. The use according to claim 15 or 16, wherein said denatured collagen is type I collagen denatured, type II collagen denatured, collagen denatured type III, denatured type IV collagen or denatured type V collagen. 18. Use in accordance with any one of the claims 15 to 17, wherein said antagonist is a antibody that has the antibody binding specificity HUI77 monoclonal (ATTC Access Number PTA-6561), HUIV26 (ATTC Access Number PTA-6563) or XL313 (ATTC Access Number PTA-6552). 19. The use according to claim 18, wherein said antibody is a humanized monoclonal antibody or chemically modified 20. Use in accordance with any one of the claims 18 or 19, wherein said antibody is a fragment of a monoclonal antibody. 21. The use according to any of claims 15 to 20, wherein said tissue is ex vivo or wherein said tissue is in vivo and said antagonist is administered intravenously, transdermally, intra-synovially, by intramuscularly, intratumorally, intraocularly, intranasally, intrathecally, topically or orally. 22. Use in accordance with any of the claims 15 to 20, wherein said antagonist is conjugated with a fluorochrome, radioactive marker, heavy metal paramagnetic, diagnostic dye or enzyme. 23. A method to select antagonists that specifically bind to a collagen or denatured collagen to inhibit angiogenesis comprising:

(to)

provide a supposed antagonist;

(b)

measure the first affinity of said supposed antagonist by a denatured collagen selected from the group consisting in type I, II, III, IV and V collagen;

(C)

measure the second affinity of said alleged antagonist by a native collagen selected from the group consisting of type I, II, III, IV and V collagen, wherein said collagen Selected native is the native form of the denatured collagen selected;

(d)

select at least one denatured collagen antagonist from the plurality of alleged antagonists that have said second affinity that is substantially less than said first affinity;

(and)

measure angiogenesis inhibition in the presence of said at less a denatured collagen antagonist; Y

(F)

select a denatured collagen antagonist that inhibits the angiogenesis

24. The method of claim 23, wherein said alleged antagonist is a non-peptide compound, preferably a small organic compound or a oligonucleotide 25. The method of claim 23, wherein said alleged antagonist is a polypeptide, a linear peptide or a cyclic peptide. 26. The method of claim 23, wherein said alleged antagonist is a monoclonal antibody or one polyclonal 27. The method of claim 23, wherein said first and said second affinity are measured by an assay enzyme-linked immunoabsorbent. 28. The method of claim 23, wherein said second affinity is approximately 3 times lower than said first affinity, preferably, said second affinity is approximately 5 times less than said first affinity, in in particular, said second affinity is approximately 10 times lower than said first affinity. 29. A method to explore antagonists of denatured collagen comprising selecting an antagonist for the ability to compete with an antagonist of the claim 13 by binding to an epitope in denatured collagen. 30. A peptide consisting of a sequence that encodes an epitope recognized by an antagonist of the claim 11, wherein said peptide is SEQ ID NO: 12.